CN110507844A - A kind of absorbable composite material and preparation method for topical acute hemostasis based on oxidizing bacteria cellulose - Google Patents

Abstract

The present invention relates to an absorbable composite material for local acute hemostasis based on oxidized bacterial cellulose and a preparation method thereof. The oxidized bacterial cellulose is prepared by using bacterial cellulose as a substrate, and then compounded with a polycationic electrolyte; or It is obtained by preparing oxidized bacterial cellulose with bacterial cellulose as a substrate, then electrostatically adsorbing and self-assembling with polycationic electrolytes, and wrapping and loading blood coagulation-promoting factors. The invention has the characteristics of acute hemostasis, broad-spectrum antibacterial, healing promotion and in vivo absorption; the preparation method is green and environmentally friendly, the compound is uniform and efficient, and the obtained composite material has high safety, and has good market application prospects in the field of advanced functional dressings.

Description

An Absorbable Compound Based on Oxidized Bacterial Cellulose for Local Acute Hemostasis Materials and their preparation methods

technical field

The invention belongs to the technical field of biomedical composite materials, in particular to an absorbable composite material for local acute hemostasis based on oxidized bacterial cellulose and a preparation method thereof.

Background technique

Absorbable hemostatic material is a kind of biomedical material that can stop bleeding quickly and can be absorbed by the human body within a certain period of time. Its biggest feature is that it is applied to local wounds in humans or animals that are difficult to suture to stop bleeding. While achieving rapid hemostasis, it can be degraded in the body without taking it out, so as to avoid secondary damage caused by removing the dressing after hemostasis is completed. At present, commonly used absorbable hemostatic materials include fibrin glue-based hemostatic materials, gelatin sponge-based hemostatic materials, oxidized cellulose-based hemostatic materials, and chitosan-based hemostatic materials. The hemostatic mechanisms and usage methods of these hemostatic materials are different, and their hemostatic effects are also very different.

Oxidized Regenerated Cellulose (ORC) was successfully prepared for the first time in 1960. It is a gauze-like or cotton-like absorbable hemostatic material made of cellulose acid after plant cellulose is oxidized after regeneration. It has the appearance and texture of cotton yarn. Soft and thin, easy to pack, apply, stuff and other operations. Its hemostatic mechanism is to gather the platelets in the surgical wound blood on the mesh gauze, use the hemostatic gauze as the hemostatic matrix, and quickly form a gel-like black substance when it encounters blood, and coagulate the blood clot to achieve the purpose of hemostasis. It does not rely on the normal blood coagulation mechanism in the human body, but rapidly promotes blood coagulation through physical action, effectively controlling bleeding in small blood vessels. At present, only the United States and the United Kingdom can produce such products, and the clinically used absorbable hemostatic gauze Surgicel is a product of Johnson & Johnson of the United States. The main component of instant yarn is oxidized regenerated cellulose. Oxidized regenerated cellulose is a derivative of cellulose obtained by highly selective oxidation of C6 primary hydroxyl groups in cellulose to carboxyl groups. It has been used in various industries, and the products sold in the market mainly rely on imports. Several companies make similar hemostatic gauzes. Although oxidized regenerated cellulose has many excellent properties, there are also some disadvantages, such as low hemostatic efficiency, not suitable for massive bleeding, weak antibacterial properties and low tissue compatibility.

At present, in some published or authorized invention patents, natural/synthetic polymer materials or inorganic materials have been used to modify oxidized regenerated cellulose to improve its antibacterial and hemostatic properties. Such as collagen (CN105079886A), alginate (CN104013991A) and carbon nanotubes (CN105056284A). Although the above composite hemostatic materials have improved the performance of oxidized regenerated cellulose to a certain extent, the improvement effect is poor, and only part of its performance can be improved, and it will have a negative impact on the bioabsorbability of the material.

Contents of the invention

The technical problem to be solved by the present invention is to provide an absorbable composite material for local acute hemostasis based on oxidized bacterial cellulose and its preparation method, which solves the problems of weak antibacterial properties of oxidized regenerated cellulose, low tissue compatibility, and hemostasis speed. slow problem.

The invention provides an absorbable composite material for local acute hemostasis based on oxidized bacterial cellulose, which is prepared by using bacterial cellulose as a substrate to prepare oxidized bacterial cellulose, and then compounding it with polycationic electrolytes; or by using bacterial cellulose The cellulose is used as a substrate to prepare oxidized bacterial cellulose, which is then self-assembled by electrostatic adsorption with a polycationic electrolyte, and the coagulation-promoting factor is packaged and loaded.

The oxidized bacterial cellulose is obtained by using bacterial cellulose as a base material, purifying with alkali and rinsing with deionized water until neutral.

The polycationic electrolyte includes one or more of chitosan, polylysine, and quaternary ammonium salts; the blood coagulation promoting factor includes one or more of thrombin and collagen.

The present invention also provides a method for preparing an absorbable composite material for local acute hemostasis based on oxidized bacterial cellulose, comprising:

(1) using bacterial cellulose as a substrate to prepare an oxidized bacterial cellulose suspension;

(2) adding the polycation electrolyte solution dropwise into the oxidized bacterial cellulose suspension to carry out electrostatic adsorption self-assembly reaction, then taking it out, rinsing, and freeze-drying to obtain a local acute hemostasis absorbable material;

Alternatively, the coagulation-promoting factor is added to the oxidized bacterial cellulose suspension for impregnation and adsorption, and then the polycation electrolyte solution is added dropwise to the oxidized bacterial cellulose suspension for electrostatic adsorption self-assembly reaction, and then taken out and rinsed , freeze-dried to obtain local acute hemostasis absorbable material.

The preparation steps of the oxidized bacterial cellulose suspension in the step (1) are as follows: the bacterial cellulose film is broken up, oxidized at room temperature by a TEMPO/NaBr/NaClO mixed oxidation system, and obtained after centrifugal cleaning and dialysis.

The polycation electrolyte solution in the step (2) is 1-5% chitosan/dilute acid solution, 0.1-1% polylysine aqueous solution or 1-10% organic silicon quaternary ammonium salt aqueous solution.

The coagulation-promoting factor in the step (2) is 0.1-10 mg/mL collagen aqueous solution or 0.01-1 mg/mL thrombin.

The mass ratio of the blood coagulation promoting factor to the oxidized bacterial cellulose in the step (2) is 1:1-10:1.

The soaking and adsorption time in the step (2) is 0.5-5 hours.

The electrostatic adsorption self-assembly reaction time in the step (2) is 1-60 minutes.

The invention uses bacterial nanocellulose as a substrate to prepare oxidized bacterial cellulose (OBC), and utilizes its high purity and large specific surface area characteristics to accelerate the coagulation process. At the same time, the opposite charge of OBC and polycationic electrolyte is used to carry out electrostatic adsorption self-assembly reaction for compounding, and blood coagulation promoting healing factors can also be introduced into it to prepare an absorbable composite material with acute hemostatic, antibacterial and healing-promoting functions.

Beneficial effect

(1) The material of the present invention uses bacterial cellulose as a substrate to prepare oxidized bacterial cellulose, which has higher carboxyl content, nanoscale fibers, larger specific surface area, lower degree of polymerization, better hemostatic effect, and better biodegradability , more easily absorbed by the body.

(2) The present invention utilizes the opposite charge of oxidized bacterial cellulose and polycationic electrolyte to carry out electrostatic adsorption self-assembly preparation without using additional cross-linking agent; at the same time, the addition of polycationic electrolyte improves the procoagulant and antibacterial properties of the material.

(3) By utilizing the large specific surface area of oxidized bacterial cellulose, the present invention conducts electrostatic adsorption self-assembly reaction with polycationic electrolytes after fully absorbing coagulation-promoting factors. and absorbable properties.

(4) The raw materials of the present invention are all green environmental protection biological materials, do not contain harmful chemical substances to the human body, can be prepared into various shapes according to actual conditions and characteristics, are safe and environmentally friendly, and can be rapidly degraded in the environment after being discarded, and can be used as a Functional materials with good performance and green environmental protection.

(5) The preparation method of the present invention is simple and easy to perform, uniform and efficient in compounding, does not use a crosslinking agent, does not cause damage to the original structure of the synthetic material, is environmentally friendly, can be industrialized, and has a good market application prospect.

Description of drawings

Fig. 1 is the schematic flow sheet of preparation method of the present invention;

Fig. 2 is the product diagram of the prepared material of embodiment 1-6; Wherein, 1 is oxidized bacterial cellulose (OBC), 2 is oxidized bacterial cellulose/chitosan (OBC/CS), 3 is oxidized bacterial cellulose/ Collagen/chitosan (OBC/COL/CS), 4 is oxidized bacterial cellulose/polylysine (OBC/PL), 5 is oxidized bacterial cellulose/organosilicon quaternary ammonium salt (OBC/SQA), 6 Oxidized bacterial cellulose/thrombin/polylysine (OBC/TB/PL);

Fig. 3 is the in vitro whole blood coagulation time test results of the materials prepared in Examples 1-6.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Example 1

(1) With Acetobacter xylinum as the strain, after static culture in liquid medium at constant temperature for 10 days, the bacterial cellulose membrane was taken out and placed in sodium hydroxide solution, treated at 80°C for 2 hours, taken out, rinsed with deionized water until Obtain bacterial cellulose film after neutralization; soak in hyaluronic acid solution for 6 hours to obtain bacterial cellulose film soaked in hyaluronic acid.

(2) Weigh 4 g of the purified bacterial cellulose membrane, shred it and disperse it in a high-speed homogenizer to become a bacterial cellulose suspension.

(3) The bacterial cellulose suspension obtained in step (2) (solid content is 1-10g/L) is oxidized at room temperature through TEMPO/NaBr/NaClO mixed oxidation system, TEMPO consumption is 0.1mmol/g, NaBr It is 1 mmol/g. A certain amount of 10% NaClO solution was added to start the reaction, and 0.5M NaOH solution was added dropwise to maintain the pH of the reaction system=10±0.5. When the NaOH solution was no longer consumed, ethanol was added to terminate the reaction. The obtained oxidized product was stored at 4°C after centrifugation, rinsing and autoclaving. The carboxyl content of the TEMPO oxidized bacterial cellulose suspension was determined to be 23% by conductometric titration.

(4) Freeze-drying the oxidized bacterial cellulose suspension obtained in step (3) to finally obtain the oxidized bacterial cellulose hemostatic material.

Example 2

(1) With Acetobacter xylinum as the strain, after static culture in liquid medium at constant temperature for 10 days, the bacterial cellulose membrane was taken out and placed in sodium hydroxide solution, treated at 80°C for 2 hours, taken out, rinsed with deionized water until Obtain bacterial cellulose film after neutralization; soak in hyaluronic acid solution for 6 hours to obtain bacterial cellulose film soaked in hyaluronic acid;

(2) Weigh 4 g of the purified bacterial cellulose membrane, shred it and disperse it in a high-speed homogenizer to become a bacterial cellulose suspension.

(3) The bacterial cellulose suspension obtained in step (2) (solid content is 1-10g/L) is oxidized at room temperature through TEMPO/NaBr/NaClO mixed oxidation system, TEMPO consumption is 0.1mmol/g, NaBr It is 1 mmol/g. A certain amount of 10% NaClO solution was added to start the reaction, and 0.5M NaOH solution was added dropwise to maintain the pH of the reaction system=10±0.5. When the NaOH solution was no longer consumed, ethanol was added to terminate the reaction. The obtained oxidized product was stored at 4°C after centrifugation, rinsing and autoclaving. The carboxyl content of the TEMPO oxidized bacterial cellulose suspension was determined to be 23% by conductometric titration.

(4) Take 2g of chitosan and fully dissolve it in 100mL of 3% acetic acid solution to form a dilute chitosan acid solution with a final concentration of 2%.

(5) Get the dilute chitosan acid solution prepared by 5mL step (4) and add dropwise in the oxidized bacterial cellulose suspension prepared by 50mL step (3), after reacting for 60 minutes, the product was repeatedly centrifuged and rinsed, and finally freeze-dried, The oxidized bacterial cellulose/chitosan composite hemostatic material was obtained.

Example 3

(1) With Acetobacter xylinum as the strain, after static culture in liquid medium at constant temperature for 10 days, the bacterial cellulose membrane was taken out and placed in sodium hydroxide solution, treated at 80°C for 2 hours, taken out, rinsed with deionized water until Obtain bacterial cellulose film after neutralization; soak in hyaluronic acid solution for 6 hours to obtain bacterial cellulose film soaked in hyaluronic acid;

(2) Weigh 4 g of the purified bacterial cellulose membrane, shred it and disperse it in a high-speed homogenizer to become a bacterial cellulose suspension.

(3) The bacterial cellulose suspension obtained in step (2) (solid content is 1-10g/L) is oxidized at room temperature through TEMPO/NaBr/NaClO mixed oxidation system, TEMPO consumption is 0.1mmol/g, NaBr It is 1 mmol/g. A certain amount of 10% NaClO solution was added to start the reaction, and 0.5M NaOH solution was added dropwise to maintain the pH of the reaction system=10±0.5. When the NaOH solution was no longer consumed, ethanol was added to terminate the reaction. The obtained oxidized product was stored at 4°C after centrifugation, rinsing and autoclaving. The carboxyl content of the TEMPO oxidized bacterial cellulose suspension was determined to be 23% by conductometric titration.

(4) Weigh 30 mg of fish-derived collagen and fully dissolve it in 10 mL of sterile water to form a protein solution with a final concentration of 3 mg/mL.

(5) Add 10 mL of the protein solution prepared in step (4) to 40 mL of the oxidized bacterial cellulose suspension prepared in step (3), stir and absorb for 1 hour to obtain the oxidized bacterial cellulose suspension with collagen adsorbed thereon.

(6) Take 2g of chitosan and fully dissolve it in 100mL of 3% acetic acid solution to form a dilute chitosan acid solution with a final concentration of 2%.

(7) get the chitosan dilute acid solution that 5mL step (6) prepares and add dropwise in the oxidized bacterial cellulose suspension that the absorption of collagen protein is prepared in 50mL step (5), after reacting for 60 minutes, the product is repeatedly centrifuged and rinsed, Finally, freeze-drying is carried out to obtain the oxidized bacterial cellulose/collagen/chitosan composite hemostatic material.

Example 4

(1) With Acetobacter xylinum as the strain, after static culture in liquid medium at constant temperature for 10 days, the bacterial cellulose membrane was taken out and placed in sodium hydroxide solution, treated at 80°C for 2 hours, taken out, rinsed with deionized water until Obtain bacterial cellulose film after neutralization; soak in hyaluronic acid solution for 6 hours to obtain bacterial cellulose film soaked in hyaluronic acid;

(2) Weigh 4 g of the purified bacterial cellulose membrane, shred it and disperse it in a high-speed homogenizer to become a bacterial cellulose suspension.

(3) The bacterial cellulose suspension obtained in step (2) (solid content is 1-10g/L) is oxidized at room temperature through TEMPO/NaBr/NaClO mixed oxidation system, TEMPO consumption is 0.1mmol/g, NaBr It is 1 mmol/g. A certain amount of 10% NaClO solution was added to start the reaction, and 0.5M NaOH solution was added dropwise to maintain the pH of the reaction system=10±0.5. When the NaOH solution was no longer consumed, ethanol was added to terminate the reaction. The obtained oxidized product was stored at 4°C after centrifugation, rinsing and autoclaving. The carboxyl content of the TEMPO oxidized bacterial cellulose suspension was determined to be 23% by conductometric titration.

(4) Weigh 0.2g chitosan and fully dissolve it in 100mL sterile water to form a polylysine aqueous solution with a final concentration of 0.2%.

(5) Add 5 mL of the polylysine aqueous solution prepared in step (4) dropwise to the oxidized bacterial cellulose suspension prepared in 50 mL of step (3), react for 60 minutes and then centrifuge and rinse the product repeatedly, and finally freeze-dry it. The oxidized bacterial cellulose/polylysine composite hemostatic material was obtained.

Example 5

(1) With Acetobacter xylinum as the strain, after static culture in liquid medium at constant temperature for 10 days, the bacterial cellulose membrane was taken out and placed in sodium hydroxide solution, treated at 80°C for 2 hours, taken out, rinsed with deionized water until Obtain bacterial cellulose film after neutralization; soak in hyaluronic acid solution for 6 hours to obtain bacterial cellulose film soaked in hyaluronic acid;

(2) Weigh 4 g of the purified bacterial cellulose membrane, shred it and disperse it in a high-speed homogenizer to become a bacterial cellulose suspension.

(3) The bacterial cellulose suspension obtained in step (2) (solid content is 1-10g/L) is oxidized at room temperature through TEMPO/NaBr/NaClO mixed oxidation system, TEMPO consumption is 0.1mmol/g, NaBr It is 1 mmol/g. A certain amount of 10% NaClO solution was added to start the reaction, and 0.5M NaOH solution was added dropwise to maintain the pH of the reaction system=10±0.5. When the NaOH solution was no longer consumed, ethanol was added to terminate the reaction. The obtained oxidized product was stored at 4°C after centrifugation, rinsing and autoclaving. The carboxyl content of the TEMPO oxidized bacterial cellulose suspension was determined to be 23% by conductometric titration.

(4) Weigh 0.1 g of the organosilicon quaternary ammonium salt and fully dissolve it in 100 mL of sterile water to form an aqueous solution of the organosilicon quaternary ammonium salt with a final concentration of 0.1%.

(5) Add 5 mL of the organic silicon quaternary ammonium salt aqueous solution prepared in step (4) dropwise to 50 mL of the oxidized bacterial cellulose suspension prepared in step (3), react for 60 minutes, and repeatedly centrifuge and rinse the product, and finally freeze-dry , to obtain the oxidized bacterial cellulose/organosilicon quaternary ammonium salt composite hemostatic material.

Example 6

(1) With Acetobacter xylinum as the strain, after static culture in liquid medium at constant temperature for 10 days, the bacterial cellulose membrane was taken out and placed in sodium hydroxide solution, treated at 80°C for 2 hours, taken out, rinsed with deionized water until Obtain bacterial cellulose film after neutralization; soak in hyaluronic acid solution for 6 hours to obtain bacterial cellulose film soaked in hyaluronic acid;

(2) Weigh 4 g of the purified bacterial cellulose membrane, shred it and disperse it in a high-speed homogenizer to become a bacterial cellulose suspension.

(3) The bacterial cellulose suspension obtained in step (2) (solid content is 1-10g/L) is oxidized at room temperature through TEMPO/NaBr/NaClO mixed oxidation system, TEMPO consumption is 0.1mmol/g, NaBr It is 1 mmol/g. A certain amount of 10% NaClO solution was added to start the reaction, and 0.5M NaOH solution was added dropwise to maintain the pH of the reaction system=10±0.5. When the NaOH solution was no longer consumed, ethanol was added to terminate the reaction. The obtained oxidized product was stored at 4°C after centrifugation, rinsing and autoclaving. The carboxyl content of the TEMPO oxidized bacterial cellulose suspension was determined to be 23% by conductometric titration.

(4) Weigh 10 mg of thrombin and fully dissolve it in 10 mL of sterile water to form a thrombin solution with a final concentration of 1 mg/mL.

(5) Take 10 mL of the thrombin solution prepared in step (4) and add it to 40 mL of the oxidized bacterial cellulose suspension prepared in step (3), fully stir and adsorb for 1 hour to obtain the oxidized bacterial cellulose suspension adsorbed with thrombin .

(6) Weigh 0.2 g of polylysine and fully dissolve it in 100 mL of sterile water to form a polylysine aqueous solution with a final concentration of 0.2%.

(7) Get 5mL of the polylysine aqueous solution prepared in step (6) and add dropwise in the oxidized bacterial cellulose suspension prepared by 50mL of step (5) to absorb thrombin, react for 60 minutes and then centrifuge and rinse the product repeatedly, Finally, freeze-drying is carried out to obtain the composite hemostatic material of oxidized bacterial cellulose/thrombin/polylysine.

The antibacterial test result of the material prepared in the embodiment 1-6 of table 1 to escherichia coli, Staphylococcus aureus and Candida albicans

Note: Sterile gauze is the control group, and the test method is based on ISO-20743-2007.

It can be seen from Table 1 that the oxidized bacterial cellulose itself does have a certain antibacterial effect, which is mainly due to the low pH environment formed by a large number of carboxyl groups in the oxidized bacterial cellulose, which inhibits the proliferation and reproduction of bacteria. However, its antibacterial effect is very limited, and it is impossible to completely avoid the infection of the wound by bacteria, especially acid-resistant bacteria and fungi. On the contrary, the antibacterial performance of the experimental group compounded with polycations was significantly improved compared with that of oxidized bacterial cellulose, and it had the same antibacterial tendency against Escherichia coli, Staphylococcus aureus and Candida albicans. The excellent broad-spectrum antibacterial properties make this series of materials have the potential for clinical application.

It can be seen from Figure 3 that the whole blood coagulation time of OBC is 248s, while the whole blood coagulation time of other composite materials is significantly lower than that of single OBC. In particular, the coagulation time of the experimental groups (Example 3 and Example 6) compounded with coagulation-promoting factors was significantly shortened, the shortest being as short as 110s. Its mechanism may be due to the synergistic effect of oxidized bacterial cellulose, polycations and coagulation-promoting factors. The invention skillfully combines these three functional materials without destroying the original structure to form a new composite material and obtain excellent hemostatic performance, which has the potential to be used clinically as an acute hemostatic material.

Claims (10)

1. a kind of absorbable composite material for topical acute hemostasis based on oxidizing bacteria cellulose, it is characterised in that: logical It crosses and prepares oxidizing bacteria cellulose by substrate of bacteria cellulose, then is compound with the progress of polycation electrolyte；Or pass through Oxidizing bacteria cellulose is prepared by substrate of bacteria cellulose, then carries out Electrostatic Absorption self assembly with polycation electrolyte, And the more factor is promoted to blood coagulation and carries out package load and obtains.

2. absorbable composite material according to claim 1, it is characterised in that: the oxidizing bacteria cellulose passes through with thin Fungin is substrate, is obtained after alkali purifies and is rinsed with deionized water to neutrality.

3. absorbable composite material according to claim 1, it is characterised in that: the polycation electrolyte includes shell One or more of glycan, polylysine, quaternary ammonium salt；Blood coagulation promotees to be cured the factor to include one of fibrin ferment, collagen Or it is several.

4. a kind of preparation method of the absorbable composite material for topical acute hemostasis based on oxidizing bacteria cellulose, packet It includes:

(1) using bacteria cellulose as substrate, oxidizing bacteria cellulose suspension is prepared；

(2) polycation electrolyte solution is added dropwise in oxidizing bacteria cellulose suspension and carries out Electrostatic Absorption from group Reaction cartridge then takes out, and rinses, and freeze-drying obtains the absorbable composite material to stop blooding for topical acute；

Alternatively, blood coagulation is promoted to be cured factor aqueous solution and be added in oxidizing bacteria cellulose suspension to impregnate absorption, then poly is positive Ionic electrolytes solution is added dropwise to progress Electrostatic Absorption self-assembling reaction in oxidizing bacteria cellulose suspension, then takes Out, it rinses, freeze-drying obtains the absorbable composite material to stop blooding for topical acute.

5. the preparation method according to claim 4, it is characterised in that: the oxidizing bacteria cellulose in the step (1) is outstanding The preparation step of turbid is as follows: bacteria cellulose film being broken up, at room temperature through TEMPO/NaBr/NaClO mixed oxidization system It is obtained after oxidation, eccentric cleaning and dialysis.

6. the preparation method according to claim 4, it is characterised in that: the polycation electrolyte in the step (2) Solution is chitosan/dilute acid soln, the polylysine aqueous solution of 0.1-1% or the organosilicon quaternary ammonium salt of 1-10% of 1-5% Aqueous solution.

7. the preparation method according to claim 4, it is characterised in that: the blood coagulation in the step (2) promotees to be cured the factor to be dense Degree is 0.1-10mg/mL collagen aqueous solution or the fibrin ferment of 0.01-1mg/mL.

8. the preparation method according to claim 4 or 7, it is characterised in that: blood coagulation in the step (2) promote to be cured the factor with The mass ratio of oxidizing bacteria cellulose is 1:1-10:1.

9. the preparation method according to claim 4, it is characterised in that: the dipping adsorption time in the step (2) is 0.5-5 hours.

10. the preparation method according to claim 4, it is characterised in that: the Electrostatic Absorption self assembly in the step (2) is anti- It is 1-60 minutes between seasonable.